2.1. Introduction
In the typical life-cycle of modern women, the mid-thirties until the cessation of menstrual periods is defined as “peri-menopause”. Women are classified as perimenopausal if menses have been experienced in the last 12 months, but with irregularity or changes in menstrual flow. During this stage of life women suffer increasing incidence of both recurrent breast pain, or “mastalgia”, and endometriosis, describing the painful condition of persistence of abnormal endometrial tissue in the abdominal cavity. These two conditions, common to the perimenopause, are poorly understood and presently lack medical therapy that is both effective and reasonably free of side effects (Prior J C, 1998, “Perimenopause: the complex endocrinology of the menopausal transition”, Endocr. Rev. 19:397-428).
While a contributing role of estrogen status has been suspected in these conditions, few consistent abnormalities in endocrine status have been identified. Circulating estrogen levels are normal in both mastalgia and endometriosis patients. There is accumulating evidence that describes diminished progesterone production during the perimenopause that may create a relative “dominance” in the activity of estrogen. However, no one theory or endocrine imbalance explains the occurrence of mastalgia and endometriosis (Santoro N et al., 1996, “Characteristics of reproductive hormonal dynamics in the perimenopause”, J. Clinical Endocrinology and Metab. 81:1495-501). Similarly, changes in estrogen to progesterone balance in women have not been associated with the occurrence of Human Papilloma Virus (HPV)-related cervical dysplasia.
2.2. Mastalgia
Recurrent, cyclical breast pain or “mastalgia” is one of the most common reasons for women's visits to their doctors. It has been estimated that 50-70% of all women experience significant mastalgia at some point in their life. In its most common form, mastalgia is a chronic condition of recurring pain, which is worse during the few days before menses (Blue J et al., 1998, “Mastalgia review: St. Marks Breast Centre”, New Zealand Medical Journal 111(1059):33-34). Traditionally, treatment choices for mastalgia have ranged from dietary manipulation (caffeine, fat, and alcohol reduction) or evening primrose oil to hormonal medications (bromocriptine and danazol) for severe breast pain. Bromocriptine (Parlodel) and danazol have a response rate of 70%, but have reported adverse side effects of up to 30-35% (Gateley C A and Mansen R E, 1991, “Management of the painful and nodular breast”, British Medical Bulletin 47:284-94; Nazli K et al., 1989, “Controlled trial of the prolactin inhibitor bromocriptine (Parlodel) in the treatment of severe cyclical mastalgia”, British Journal of Clinical Practice 43(9):322-7; Kontosolis K et al., 1997, “Comparison of tamoxifen with danazol for treatment of cyclical mastalgia”, Gynecol. Endocrinol. 11:393-397). The use of medroxyprogesterone acetate to support levels of progesterone, possibly low in this condition, provided relief no better than placebo in a controlled trial (Maddox P R et al., 1990, “A randomized controlled trial of medroxyprogesterone acetate in mastalgia”, Annals of the Royal College of Surgeons of England 72(2):71-6).
The approach of dietary supplementation for mastalgia has been explored by earlier investigators. This included the addition of high doses of evening primrose oil, beta carotene, and vitamin A to the diet of affected women. Evening primrose oil is used by British physicians as an initial intervention to control mastalgia because of its non-hormonal composition. Though it has been found to normalize the ratio of essential fatty acids to saturated fatty acids in the serum of women with mastalgia, the therapy requires 3 to 4 months for benefit. Improvements were seen in up to 40% of patients but side effects included bloating and nausea (Maddox P R, 1989, “The management of mastalgia in the UK”, Hormone Research 32:21-27). Italian researchers explored the addition of combinations of beta carotene and Vitamin A (retinol) in the management of mastalgia. (Santamaria L et al., 1989, “Beta-carotene supplementation associated with intermittent retinol administration in the treatment of pre-menopausal mastodynia”, Boll Chim Far 128:284-287). Some success was reported, but the high doses of retinol required (150-300,000 I.U per day) are in the range associated with significant side effects which include headache, skin lip and mouth dryness, nausea, dizziness, and alopecia. Based on the common occurrence of mastalgia as a disorder in women, the need exists for more effective therapy with acceptable risks and side effects (Ashley B, 1998, “Mastalgia”, Lippincotts Primary Care Practice 2(2):189-93).
2.3. Endometriosis
Endometriosis is a disease that affects as many as 15% of fertile women and up to 50% of infertile women. Its occurrence increases with age and is greatest in the perimenopausal years (Tzingounis V A and Cardamakis E, 1997, “Modem approach to endometriosis”, Annals of the New York Academy of Sciences 816:320-330). Endometriosis refers to the presence of functional endometrial glands and stroma in abnormal locations outside the uterine cavity. Despite extensive research, the natural history and pathogenesis of endometriosis is still poorly understood and remains controversial. As with mastalgia, most therapeutic approaches have been directed at hormonal therapy. The most common therapy involves the use of Danazol. Danazol is a synthetic steroid with androgenic action suppressing the pituitary gland cycling necessary for menstrual periods. Amenorrhea, or lack of menstrual periods results. Though providing some relief from the pain of endometriosis adverse side effects are experienced in up to 80% of women using Danazol (Greenblatt R B et al., 1971, “Clinical studies with an anti-gonadotropin—danazol”, Fertil Steril 22:102). Notably these side effects include weight gain, fluid retention, acne, decreased breast size, hot flushes and mood changes. In addition to Danazol, other hormonal manipulations used in the management of endometriosis involve use of gonadotropin releasing hormone analogues (GnRH) and the drug gestrinone, a synthetic steroid derived from 19-nortestosterone. The side effects associated with these therapies are significant and include the spectrum of symptoms associated with hypoestrogenism and menopause. These include hot flushes, night sweats, and osteoporosis (Telimaa E J et al., 1987, “Placebo-controlled comparison of danazol and high-dose medroxyprogesterone acetate in the treatment of endometriosis”, Gynecol Endocrinol 1:51; Thomas E. J. et al., 1987, “Impact of gestrinone on the course of asymptomatic endometriosis”, Br. Med J., 294:272). Clearly, more benign approaches to the management of the pain of endometriosis are needed.
2.4. Cervical Dysplasia
Cervical dysplasia, also known as cervical intraepithelial neoplasia (CIN), refers to the occurrence of abnormal cells and tissue involving the surface of the uterine cervix, which consists of the lowest part of the uterus. Anatomically, the cervix provides the portal of entry from the vagina to the interior of the uterus. Its surface consists of an epithelial “transition-zone” where superficial, flattened cells characteristic of vaginal mucosa abruptly change to cuboidal epithelial cells characteristic of the interior surface of the uterus (endometrium). The designations CIN I, II and III refer to mild, moderate, and severe dysplasia/carcinoma in situ (CIS) of the cervical epithelium, respectively. This histological grading scheme is based largely on the extent to which the thickness of the epithelium is replaced by abnormal, mitotically active cells with enlarged, hyperchromatic nuclei (Ferenczy A et al., “Cervical Intraepithelial Neoplasia and Condyloma”, In R. J. Kurman, Editor. Blaustein's Pathology of the Female Genital Tract, Springer-Verlag, 1987). Untreated, a subset of dysplastic lesions will advance to cervical cancer, with a frequency that increases dramatically in CIN III lesions (Ostor A G, 1993, Int. J. Gynecol. Path. 12:186-192).
In many developing countries, cervical cancer is the most common cancer (excluding skin) in women and the major cause of cancer-related deaths in women (Parkin D et al., 1993, Int J Cancer 54:594-606). In the United States, abnormalities on Papanicolaou, or “Pap” smears are detected in millions of women annually, resulting in an estimated annual cost of $6 billion for patient evaluation and treatment (Kurman R et al., 1994, JAMA 271:1866-9).
Cervical dysplasia in women is a common condition that is linked to infection by HPV (Walboomers J M et al., 1999, “Human papillomavirus is a necessary cause of invasive cervical cancer worldwide”, J Pathol. 189(1):12-9). While dependent on the presence of HPV, the occurrence of cervical dysplasia in women has not been related to estrogen or progesterone levels. Regarding estrogen metabolism, the severity of cervical dyplasia, documented by stage of CIN on biopsy, has been associated with a reduced 2-hydroxy metabolism of estrogen in some women (Sepkovic et al., 1995, “Estrogen Metabolite Ratios and Risk Assessment of Hormone-related Cancers”, Annals New York Academy of Sciences 768:312-316).
The most common methods used to treat cervical dysplasia include removal or eradication of the abnormal tissue, using methods such as cryosurgery, laser vaporization, and surgical removal. Due to the potential for harm to normal tissue, and recurrence of dysplasia following surgery, a need clearly exists for more effective and less invasive therapy.
2.5. Dietary Indoles
3,3′-Diindolylmethane (DIM), as well as its precursor, Indole-3-carbinol (I3C), and cogener, 2-(Indol-3-ylmethyl)-3,3′-diindolylmethane (LTr-1), are natural phytochemicals and part of the family of dietary indoles discovered in cruciferous vegetables. DIM and I3C are found in cruciferous vegetables including broccoli, cauliflower, cabbage and Brussels sprouts (Bradfield C A and Bjeldanes L F, 1987, “High performance liquid chromatographic analysis of anticarcinogenic indoles in Brassica oleracea”, J. Agric. Food Chem. 35:46-49). DIM, together with the linear trimer, LTr-1, are formed after the enzymatic release of I3C from parent glucosinolates found in all cruciferous vegetables.
It is now known that there is a connection between dietary cruciferous indoles and estrogen metabolism. H. Leon Bradlow, Ph.D. and his group at the Strang Cancer Prevention Laboratory in New York were the first to establish the link between phytonutrients from cruciferous vegetables and estrogen metabolism. They showed that supplemental use of I3C can act to promote a dramatic change in the metabolism of estrogen (Michnovicz J J et al., 1997, “Changes in levels of urinary estrogen metabolites after oral indole-3-carbinol treatment in humans”, J Natl Cancer Inst. 89(10):718-23). This change in metabolism has the power to greatly reduce estrogen exposure as a risk for cancer and provides a dietary approach to improving estrogen metabolism. I3C has been shown to increase 2-hydroxy estrogen metabolism in women (Bradlow H L et al., 1994, “Long-term responses of women to indole-3-carbinol or a high fiber diet”, Cancer Epidemiol Biomarkers Prev. 3(7):591-5). When cruciferous phytochemicals are added to the diet, estrogen metabolism is shifted. This produces a predominance of 2-hydroxy and 2-methoxyestrogens (Michnovicz J J et al., 1997, “Changes in levels of urinary estrogen metabolites after oral indole-3-carbinol treatment in humans”, J Natl Cancer Inst. 89(10):718-23). An increased proportion of 2-hydroxy metabolites is correlated to protection from breast cancer. This relationship has been documented in case-control studies (Ho G H et al., 1998, “Urinary 2/16 alpha-hydroxyestrone ratio: correlation with serum insulin-like growth factor binding protein-3 and a potential biomarker of breast cancer risk”, Ann Acad Med Singapore 27:294-299; Schneider J. et al., 1982, “Abnormal oxidative metabolism of estradiol in women with breast cancer”, Proc Natl Acad Sci USA 79:3047-3051). The 2-hydroxy metabolites have been called “good estrogens” (Bradlow H L et al., 1996, “2-hydroxyestrone: the ‘good’ estrogen”, J Endocrinol. 150 Suppl:S259-65), and may function as antioxidants (Komura S et al., 1996, “Catecholestrogen as a natural antioxidant”, Ann N Y Acad Sci. 786:419-429).
With regard to prior art and the dietary indoles, the supplemental use of I3C, which converts in part to DIM, LTr-1, and other compounds after passage through the stomach, has been the subject of U.S. Pat. No. 5,895,787, describing the use of I3C and related dietary indoles to reduce the symptoms of fibromyalgia. Despite this use, no relationship between fibromyalgia and estrogen status has been documented (Bengtsson A. and Henriksson, K. G., 1986, “Primary fibromyalgia. A clinical and laboratory study of 55 patients”, Scand J. Rheumatol 15(3):340-7). Published reports have demonstrated some clinical improvement following dietary supplementation with I3C in recurrent laryngeal papillomatosis and cervical dysplasia (Rosen C A et al., 1998, “Preliminary results of the use of indole-3-carbinol for recurrent respiratory papillomatosis”, Otolaryngology Head Neck Surgery 118: 810-5; Jin L. et al., 1999, “Indole-3-carbinol prevents cervical cancer in human papilloma virus type 16 (HPV16) transgenic mice”, Cancer Res. 59(16):3991-7; Baugh S M et al., 1998, “Treatment of cervical dysplasia with indole-3-carbinol”, Ray A. Barlow Scientific Symposium, Louisiana State University Medical School, Shreveport, La.; Bell M C et al., 2000, “Placebo-controlled trial of indole-3-carbinol in the treatment of CIN”, Gynecol Oncol. 78(2):123-9). These diseases are both conditions related to the presence of the human papilloma virus. Reports of adverse side effects with I3C use in animals and in at higher doses in individuals with respiratory HPV infection (Recurrent Respiratory Papillomatosis [“RRP”]) have discouraged further clinical testing of I3C (Rosen C A et al., 1998, “Preliminary results of the use of indole-3-carbinol for recurrent respiratory papillomatosis”, Otolaryngology Head Neck Surgery 118:810-5; Wilker C et al., 1996, “Effects of developmental exposure to indole-3-carbinol or 2,3,7,8-tetrachlorodibenzo-p-dioxin on reproductive potential of male rat offspring”, Toxicol Appl Pharmacol. 141(1):68-75). I3C's use is associated with a number of safety concerns in reproductive-age women due to its enzyme-inducing actions and developmental toxicity.
Thus, the individual metabolic products of I3C are being investigated to discover safer and more effective alternatives to I3C. Following oral administration of I3C, DIM is but one of many derivatives generated from I3C. I3C is highly reactive in stomach acid, which leads to the production of a large number of biologically active byproducts distinct from DIM. DIM arises from I3C as one of the reaction products in more neutral pH environments, not typically seen in the gastric environment. I3C exposure to acid produces many non-DIM products, including indolo(3,2,b)carbazole (ICZ), and the cyclic trimer, 5,6,11,12,17,18-hex-hydrocyclononal[1,2-b:4,5-b′:7,8-b]:triindole, and others which are more highly enzyme inductive than DIM (De Kruif C A et al., 1991, “Structure elucidation of acid reaction products of indole-3-carbinol: detection in vivo and enzyme induction in vitro”, Chem Biol Interact 80(3):303-15).
DIM differs from I3C in its structure and in its chemical and biological properties. Unlike I3C, DIM is an acid-stable indole with less influence on carcinogen metabolism than I3C and non-DIM products from I3C (Bradfield C A and Bjeldanes L F, 1987, “Structure-activity relationships of dietary indoles: a proposed mechanism of action as modifiers of xenobiotic metabolism”, J Toxicol Environ Health 21(3):311-23). Unlike I3C, DIM has been shown to influence estrogen metabolism in animals without the need for intra-gastric conversion into other indole reaction products (Jellinck P H et al., 1993, “Ah receptor binding properties of indole carbinols and induction of hepatic estradiol hydroxylation”, Biochem Pharmacol 45:1129-1136).
In cell culture, DIM has been shown to have both growth promoting and growth inhibitory properties. In MCF7 breast cancer cells, DIM promoted growth when tested in the absence of estrogen (Riby J E et al., 2000, “Ligand-independent activation of estrogen receptor function by 3,3′-diindolylmethane in human breast cancer cells”, Biochem Pharmacol 60(2):167-77). When tested in colon cancer cells, DIM demonstrated selective growth inhibitory activity which was not observed in non-cancerous colon cells (Gamet-Payrastre L et al., 1998, “Selective cytostatic and cytotoxic effects of glucosinolates hydrolysis products on human colon cancer cells in vitro”, Anti-Cancer Drugs 9:141-148).
In another study, oral DIM was shown to impede the growth of carcinogen-induced breast cancer in animals (Chen I. et al., 1998, “Aryl hydrocarbon receptor-mediated antiestrogenic and antitumorigenic activity of diindolylmethane”, Carcinogenesis 19(9):1631-9).
There have been no reports on the usefulness of DIM or LTr-1 in the treatment or prevention of mastalgia, endometriosis or HPV-related conditions, including cervical dysplasia. Isolation and formulation requirements for DIM as a pure substance limited its availability for animal experiments. Pure, isolated DIM was not available for human use prior to Summer, 1998. Use of DIM and LTr-1 in absorption enhancing formulations for improving the balance of estrogen metabolites has been the subject of earlier investigation by the present inventor and provides the basis of U.S. Pat. No. 6,086,915. This work has allowed for the present investigation of the use of dietary indoles as dietary supplements to beneficially impact mastalgia, endometriosis, and cervical dysplasia.
Therefore, safer and more consistently effective treatments are needed for mastalgia, endometriosis and HPV-related conditions, including cervical dysplasia.